CA2264961A1 - Apparatus and method for performing sequencing of nucleic acid polymers - Google Patents

Abstract

An apparatus for processing samples containing DNA to produce a sequencing fragment mixture comprises a sample processing element comprising: a thermocycling region having one or more chambers for receiving a DNA sequencing reaction mixture and forming sequencing fragments therefrom; a separation region comprising a separation matrix for separating the sequencing fragments formed in the thermocycling regions; a detection region for detection of the separated sequencing fragments; and means for regulating the temperature in the thermocycling region of the sample processing element to provide a plurality of thermal cycles, each cycle including at least a denaturation phase and an extension phase. The apparatus for processing sample can be placed in a holder which is associated with means for applying an electric field to the separation region of a sample processing apparatus placed within the holder to cause polynucleotide sequencing fragments to migrate through the separation region from the thermocycling region to the detection region; and means for detecting polynucleotide fragments within the detection region of the sample processing apparatus placed within the holder.

Description

10152025CA 02264961 1999-02-26WO 98/08978 PCT/US97/ 15056_1_DESCRIPTIONAPPARATUS AND METHOD FOR PERFORMING SEQUENCINGOF NUCLEIC ACID POLYMERSBACKGROUND OF THE INVENTIONThis application relates to apparatus for performing DNA sequencing reactions, and inparticular to improved apparatus for performing sequencing reaction Protocols making use ofthermally stable polymerase enzymes having enhanced capacity to incorporate chainterminating nucleotides during chain termination sequencing reactions.DNA sequencing is generally performed using techniques based on the "chaintermination” method described by Sanger et al., Proc. Nat’! Acad. Sci. (USA) 74 (12):5463-5467 (1977) . Basically, in this process, DNA to be tested is isolated, rendered singlestranded, and placed into four vessels. In each vessel are the necessary components toreplicate the DNA strand, i.e., a template-dependant DNA polymerase, a short primer mole-cule complementary to a known region of the DNA to be sequenced, and the standarddeoxynucleotide triphosphates (dNTP’s) commonly represented by A, C, G and T, in a bufferconducive to hybridization between the primer and the DNA to be sequenced and chainextension of the hybridized primer. In addition, each vessel contains a small quantity of onetype (i.e., one species) of dideoxynucleotide triphosphate (ddNTP), e.g. dideoxyadenosinetriphosphate (ddA).In each vessel, the primer hybridizes to a specific complementary site on the isolatedDNA. The primers are then extended, one base at a time to form a new nucleic acid polymercomplementary to the isolated pieces of DNA. When a dideoxynucleotide triphosphate isincorporated into the extending polymer, this terminates the polymer strand and prevents itfrom being further extended. Accordingly, in each vessel, a set of extended polymers ofspecific lengths are formed which are indicative of the positions of the nucleotidecorresponding to the dideoxynucleotide in that vessel. These sets of polymers are thenevaluated using gel electrophoresis to detennine the sequence.Improvements to the original technique described by Sanger et al. have includedimprovements to the enzyme used to extend the primer chain. For example, Tabor et al. havedescribed enzymes such as T7 DNA polymerase which have increased processivity, and1015202530WO 98/08978CA 02264961 1999-02-26PCTIUS97/15056-2-increased levels of incorporation of dideoxynucleotides. (See US Patent No. 4,795,699 andEP-A-0 386 857, which are incorporated herein by reference). More recently, Reeve et al.have described a thermostable enzyme preparation, called ThermoSequenaseTM, withimproved qualities for DNA sequencing. Nature 376: 796-797 (1995); EP-A-0 655 506,which is incorporated herein by reference. For sequencing, the ThermoSequenase”" productis used with an amplified DNA sample containing 0.5-2 u g of single stranded DNA (or 0.5 to5 ti g of double stranded DNA) into four aliquots, and combining each aliquot with theThermoSequenaseTM enzyme preparation, one dideoxynucleotide termination mixturecontaining one ddNTP and all four dNTP's; and one dye-labeled primer which will hybridize tothe DNA to be sequenced. The mixture is placed in a thermocycler and run for 20-30 cyclesof annealing, extension and denaturation to produce measurable amounts of dye-labeledextension products of varying lengths which are then evaluated by gel electrophoresis. EP-A-0 655 506 further asserts that ThermoSequenaseTM and similar enzymes can be used foramplification reactions.Each of the processes known for determining the sequence of DNA can be precededby amplification of a selected portion of the genetic material in a sample to enrich the concen-tration of a region of interest relative to other DNA. For example, it is possible to amplify aselected portion of a gene using a polymerase chain reaction (PCR) as described in U.S.Patents Nos. 4,683,194, 4,683,195 and 4,683,202, which are incorporated herein byreference. This process involves the use of pairs of primers, one for each strand of the duplexDNA, that will hybridize at a site located near a region of interest in a gene. Chain extensionpolymerization (without a chain terminating nucleotide) is then carried out in repetitive cyclesto increase the number of copies of the region of interest many times. The amplified poly-nucleotides are then separated from the reaction mixture and used as the starting sample forthe sequencing reaction. Gelfand et al. have described a thermostable enzyme, "Taqpolymerase," derived from the organism T hermus aquaticus, which is useful in thisamplification process. (See US Patent Nos. 5,352,600 and 5,079,352 which are incorporatedherein by reference).Ruano and Kidd, Proc. Nat '1. Acad. Sci. (USA) 88: 2815-2819 (1991) and U.S. PatentNo. 5,427,91 1, which are incorporated herein by reference, describe a process which they call“coupled amplification and sequencing” (CAS) for sequencing of DNA. In this process, agf"T"’_l0l525“i“"‘°lL9P5':N CA 02264961 1999-02-26-3-Ruano and Kidd, Proc. Nat ’Z. Acad. Sci. (USA) 88: 2815-2819 (1991) and U.S. PatentNo. 5,427,911, which are incorporated herein by reference, describe a process which they call“coupled amplification and sequencing” (CAS) for sequencing ofDNA. In this process, asample is treated in a first reaction stage with two primers and amplified for a number ofcycles to achieve 10,000 to 100,000-fold amplification. A dd.NTP is then added during theexponential phase of the amplification reaction, and the reaction is processed for additionalthermal cycles to produce chain-terminated sequencing fragments. The CAS process doesnot achieve the criteria set forth above for an ideal diagnostic assay because it requires anintermediate addition of reagents (the ddNTP reagents). This introduces and opportunity forerror or contamination and increases the complexity of any apparatus which would be usedfor automation.While the methods now available for DNA sequencing produce useful results, they allinvolve multiple steps and are carried out in multiple pieces of apparatus usually including atleast a thermocycling apparatus for performing an initial amplification, an apparatus for per-forming a sequencing reaction, and an electrophoresis apparatus for separating the sequencingreaction products. In some cases, the detection of the sequencing reaction products is per-formed in real time, and the detection system is incorporated as part of the electrophoresisapparatus. In others, the detection of the sequencing fragments is performed after theseparation is completed using a further piece of apparatus. While this use of multiple piecesof apparatus is reasonably well-suited for use in a research environment, where the sequenceof genetic materials is being determined for the first time, it is less well-suited for use in aroutine diagnostic procedure wherein the sequence of the same region of DNA is determinedover and over again in multiple patients. For this latter purpose, it would be desirable to havea single apparatus which could perform the complete processing of a complex DNA sample,for example genomic or other natural abundance DNA. It is the object of the presentinvention to provide such an apparatus and method.EP-A2-O 572 023 discloses an electrophoretic apparatus for determining thebase sequence of DNA. The apparatus has a device for introducing sample into anelectrophoresis gel, a device for controlling the temperature of sample in the sample-AMENDED SHEETF?CM :CA 02264961 1999-02-26 FFCME NC. I 3iJCFPEDQHLZLQRSCN10.3A..introducing device, a device for causing electrophoretic migration within the electrophoresisgel, and a device for analyzing separated fragments.W093-22058 discloses a thermal cycling device in which DNA to beamplified is pumped back-and-forth between chambers at 94°C and 65°C and then into ananalysis chamber where a sequence-specific nucleic acid hybridization assay is performed. Ithas no disclosure of use of the invention for either DNA sequencing or in conjunction with anelectrophoresis apparatus.SUMMARY OF THE INVENTIONThe present invention provides an apparatus and associated sample processingelement for performing sequencing of a DNA containing sample, particularly a sample ofgenomic or other natural abundance DNA. The apparatus comprises(a) a holder for receiving a sample processing element having a thermocyclingregion, a separation region and a detection region;AMENDED SHEET1015202530CA 02264961 1999-02-26WO 98/08978 PCT/U S97/ 15056-4-(b) means for regulating the temperature within the thermocycling region of asample processing element placed within the holder;(c) means for applying an electric field to the separation region of a sampleprocessing element placed within the holder to cause polynucleotide fragments formed in thethermocycling region to migrate through the separation region from the thermocycling regionto the detection region; and((1) means for detecting polynucleotide fragments within the detection region ofa sample processing element placed within the holder.Sequencing is performed by loading a genomic or natural abundance DNA-containingsample; a thermostable polymerase such as ThermoSequenaseTM which incorporatesdideoxynucleotides into an extending nucleic acid polymer at a rate which is no less than about0.4 times the rate of incorporation of deoxynucleotides; two primers which bind tocomplementary strands of a target DNA molecule at sites flanking a region of interest; amixture of nucleotide triphosphates (A, C, G and T) and one dideoxynucleotide triphosphateinto the thermocycling region of a sample processing element placed within the apparatus, andprocessing the combination through multiple cycles of annealing, extension and denaturationto form a mixture of sequencing fragments within the thermocycling region. An electric fieldis then applied to the sample processing element to cause the sequencing fragments to migratefrom the thermocycling region, through the sample processing element and to the detectionregion. As the fragments pass through the detection region, they are detected and the outputsignal is analyzed to yield the sequence of the region of interest within the target sequence.BRIEF DESCRIPTION OF THE DRAWINGSFigs. 1A and 1B show an embodiment of a sample processing element and apparatus inaccordance with the invention;Fig. 2 shows a further embodiment of the apparatus of the invention;Fig. 3 shows a section view through the thermocycling region of an embodiment of asample processing element in accordance with the invention;Fig. 4 shows an embodiment of an apparatus of the invention;Fig. 5 shows an embodiment of an apparatus of the invention;l015202530CA 02264961 1999-02-26WO 98/08978 PCT/U S97/ 15056-5-Fig. 6 shows a further embodiment of a sample processing element in accordance withthe invention;Fig. 7 shows a thermocycling and concentration chamber useful as part of a sampleprocessing element in accordance with the invention;Fig. 8 shows a further embodiment of the invention;Fig. 9 shows an embodiment of a thermocycling chamber useful in the presentinvention;Fig. 10 shows the positioning of a denaturing buffering in an electrophoresis gelforming part of a sample processing element of the invention;Figs 11A and 11B show sequencing fragments patterns obtained usingThermoSequenaseT'V' or Vent/Sequitherm; andFig. 12 shows a sequencing fragment pattern obtained using ThermoSequenaseTM.DETAILED DESCRIPTION OF THE INVENTIONThe present invention provides an apparatus which helps to exploit the properties ofenzymes like ThermoSequenaseTM, namely the ability to incorporate dideoxynucleotides intoan extending polynucleotide at a rate which is no less than about 0.4 times the rate ofincorporation of deoxynucleotides, to enable sequencing of a nucleic acid polymer from asample in a single apparatus. Although the apparatus and sample processing element of theinvention can be used to analyze any DNA containing sample including samples which havebeen previously amplified, they are particularly suited for analysis of samples containingnatural abundance DNA. As used herein a “natural abundance sample” is a sample which hasbeen treated to make DNA in the sample accessible for hybridization with oligonucleotideprimers, for example by lysis, centrifugation to remove cellular debris and proteolytic digestionto expose the DNA, but which has not been subjected to a preferential purification oramplification step to increase the amount of target DNA relative to non—target DNA present inthe initial sample. The term “natural abundance” does not, however, require the presence ofall the DNA from the original sample. Thus, a complex sample containing just nuclear DNA,or just mitochondrial DNA or some subfraction of nuclear or mitochondrial DNA obtained byisolation from a tissue sample but not subjected to preferential amplification would be a“natural abundance” sample within the meaning of that term in the specification and claims of1015202530CA 02264961 1999-02-26WO 98/08978 PCTIUS97/15056-6-this application. The term “natural abundance” would also include a DNA sample prepared byconversion, for example by reverse transcription, of a total mRNA preparation or the genomeof an RNA virus to cDNA; DNA isolated from an individual bacterial colony growing on aplate or from an enriched bacterial culture; and a viral DNA preparation where substantiallythe entire viral genome is isolated. The term “natural abundance” does not encompass asample in which the isolated DNA is not a complex combination of DNA molecules, and thuswould not encompass, for example, a purified plasmid preparation containing only a singlespecies of plasmid.A first embodiment of the apparatus of the present invention comprises four basicelements:(a) a holder for receiving a sample processing element having a thermocyclingregion, a separation region and a detection region;(b) means for regulating the temperature within the thermocycling region of asample processing element placed within the holder;(c) means for applying an electric field to the separation region of a sampleprocessing element placed within the holder to cause polynucleotide fragments formed in thethermocycling region to migrate through the separation region from the thermocycling regionto the detection region; and(d) means for detecting polynucleotide fragments within the detection region ofa sample processing element placed within the holder.The sample processing element of the present invention can be considered as threefunctional regions: a thermocycling region, a separation region and a detection region. As willbe apparent from the various embodiments discussed below, these regions can be parts ofintegrated device, or can be separate component parts.In general, the thermocycling region is a chamber in which the chemical reactantsnecessary for forming sequencing fragments directly from DNA are placed and exposed tocycles of temperature effective to promote denaturation and annealing/extension. This can beachieved by varying the temperature of the entire thermocycling region or by creating discretetemperature bands within the region.The separation region of the sample processing element can be any type of separationmatrix that is effective to separate DNA sequencing fragments on the basis of fragment size. ‘.0152.02530CA 02264961 1999-02-26Thus, while the examples below refer to separation regions made from electrophoresis gels. itwill be understood that other types of separation matrices, including the separation matricesdescribed in International Patent Publication Nos. W096/42012 and W096/42013 filed June7, 1996 and incorporated herein by reference. may also be used. The detection region of thesample processing element may be a contiguous part of the separation region, distinguishableonly by the fact that detection of separated fragments occurs in this region. The detectionregion may also be a discrete part of the sample processing element, however, in whichfragments are detected after leaving the separation region.Figs. 1A and 1B show a first embodiment of the sample processing element andapparatus of the present invention. The sample processing element is a separation matrix suchas an electrophoresis gel 10, optionally supported by a substrate 11. The electrophoresis gel10 has three functional regions: a therrnocycling region 101, a separation region 102 and adetection region 103. Within the therrnocycling region 101 are a plurality of wells 12 intowhich the reaction mixtures for the production of sequencing fragments are placed.A sample processing element formed from a polyacrylamide gel 10 and substrate 11are placed within a holder 13 of an apparatus as shown in Fig. 1B. The holder 13 positionsthe polyacrylarnide gel 10 such that the wells 12 are in alignment with a temperature regu-lating element, for example a Peltier heating and cooling device 14 powered via leads 114 and114’. The holder 13 also positions the sample processing element with respect to electrodes15 and 16 which are used to generate the electrophoretic field with the polyacrylamide gel 10,and places the detection zone 103 of the sample processing element in alignment with a lightsource 17 for supplying excitation suitable for excitation Of fluorescent labels on the sequen-cing fragments and an array of detectors 18 for detecting emission from the fluorescent labels.Regulating the temperature of the thermal cycling region can be achieved by a varietyof methods. One method, illustrated in Fig. 1B, shows the use of a Peltier device 14 for heat-ing and cooling. These devices can maintain a temperature of 0°C to 100°C within +/- 0.5°C. A limitation on these devices is that the speed of cooling may result in a requirement for apotentially significant period of time in which to effect the transition between the tempera-tures. These temperature changes can be quite large, for example from a 94°C denaturationtemperature to a 45°C annealing temperature. This long time period is useable for someapplications, but in others it will lead to the appearance of spurious bands and false priming.AMENDED SHEET1015202530CA 02264961 1999-02-26WO 98/08978 PCT/U S97/ 15056-8-A preferred apparatus employs a relatively large cooling sink 20 in the thermal cyclingregion 101 as shown in Fig. 2. The sink temperature is maintained below the minimumcycling temperature, at 4 to 35 ° C, preferably about 25 °C. Reagents are loaded into the wells12 within the thermocycling region of the sample processing element, where they begin toequilibrate to the temperature of the sink 20. To begin the reaction, a radiation source 21directed to the therrnocycling zone 101 is switched on. The radiation 210 may be microwave,visible light, infrared radiation or any other radiation that can be absorbed by the sample andthat will not substantially damage the reactants. The radiation is applied to increase thesample temperature to the desired temperature (i.e. 94°C for denaturation) . At the end ofthe desired time period, the radiation intensity is changed to produce the next desiredtemperature in the sample. The large cooling sink 20 rapidly reduces the temperaturewhenever the radiation source 21 is turned off, thus providing a rapid transition for thesample. The radiation intensity therefore determines the sample temperature.The temperature of reagents with the thermocycling region 101, should be maintainedwithin approximately +/- 0.5°C of the desired temperature. The control of the radiationintensity therefore requires careful consideration. A variety of detectors may be used todetermine the sample temperature. Temperature sensitive films using liquid crystals (EdmundScientific Co., Barrington, NJ) can determine temperature to within +/- 0.1 °C. These filmscould be placed beside or underneath the sample wells 12, and so provide a precise method oftemperature detection. Alternatively, since the refractive index of the sample will change withtemperature, detectors of refractive index may be employed. Further options includetemperature sensitive dyes added directly to the sample. Detection of electrical capacitancewhich changes with the temperature of the solution can also be used. In all the above cases,the detectors can be linked to microprocessors which change the intensity of the radiationsource to obtain the desired temperature.The apparatus of the invention also includes means for applying an electric field to theseparation region of the sample processing element. This can be in the form of solutionelectrodes, disposed at either end of the sample processing element. The electrophoresis gelwithin the sample processing element is immersed in a buffer in two wells, each of whichcontains an electrode which is connected to a power supply for generating the electrophoreticfield. Alternatively, the electrodes can be printed on the surface of the sample processing"55 ":015.202530CA 0226496i_1999—02—26-9-element. Printed electrodes can be formed from a variety of materials. including indium tinoxide (ITO) or platinum, In either case. an electric field is generated between the electrodes ofsufficient magnitude to cause the sequencing fragment to migrate from through the separationregion 102 where they are separated into bands based upon the size the fragment.Once the sequencing fragments are separated by electrophoresis through the separationregion 102 of the sample processing element, they are detected in the detection region 103.The type of detection system employed will depend on the type of label incorporated into thesequencing fragments. The preferred type of label will be a fluorescent label, in which casedetection of sequencing fragments can be achieved using fluorescence detection means, asdescribed for example in US Patent No. 5,710,628, which is incorporated herein by reference.Such a detection scheme is shown generally in Fig. 1B, in which a radiation source 17provides excitation energy to fluorophores in the sequencing fragments which are detected byan array of fluorescence detectors 18. It will be appreciated that various types of light sourcesproducing light of an appropriate wavelength for excitation of the fluorophores may beemployed, including lasers, laser diodes, and light-ernitting diodes. The light may be split intoindividual excitation bearnlets for excitation of multiple detection sites (corresponding to thelanes of the gel) within the detection region using optical fibers, diffractions gratings, a spotarray generation grating or other optical components. Alternatively, a plurality of lightsources, one for each detection site can be used. In addition, for multi-dye applications theapparatus may provide light of several different wavelengths to the detection site, eitherthrough the use of multiple light sources or using optical filters.The array of detectors 18 may provide a separate detector for each detection site withinthe detection region 103, or one detector may be aligned to collect light from several adjacentdetection site. In the latter case, the excitation light beams to the adjacent sites are suitablyapplied to a temporally staggered fashion so that emission from the detection sites can bedistinguished.While fluorescence labeling and detection is the preferred method of practicing theinvention, other types of labeling and detection can be used as well. For example, achromophore or a chromogenic label can be used with a photometric detection system, or astrongly chiral label could be used with a polarization detection system as described in USPatent Application Serial No. 08/387,272, which is incorporated herein by reference.AMENDED SHEET1015202530CA 02264961 1999-02-26WO 98/08978 PCT/U S97/ 15056-10-The output of the detector array is an electrical signal representing the positionof one more bases in the target sequence. This signal is preferably transferred to a dataprocessing apparatus, such as a micro or minicomputer for data alignment and base calling.Data alignment and base calling is preferably performed using the techniques described in USPatent Applications Nos. 08/497,202 and 08/670,534 which are incorporated herein byreference.A further aspect of the present invention is the sample processing element which isplaced within the apparatus of the invention. This sample processing element is, as notedabove, divisible into three functional regions: a thermocycling region, a separation region anda detection region. These regions are contiguous one with the other, and are arranged so thatsequencing fragments produced in the thermocycling region are electrophoresed through theseparation region to form discrete ,bands of polynucleotides which are detected in thedetection region.The basic component of the sample processing element of the invention is apolyacrylamide gel 10, optionally supported by a substrate 11 or a pair of substrates. In theembodiment shown in Fig.lA, the thermocycling region 101 is different from the separationand detection regions 102 and 103 (which are structurally indistinguishable from one another)by virtue of the wells 12 formed in the polyacrylamide gel to receive the sequencing reactionmixture. The wells need to hold this reaction mixture with sufficient integrity for the durationof the thermal cycling reaction. This may take up to 2 hours in some cases, althoughpreferably it would be completed in under 30 minutes.If a polyacrylamide gel slot is not a satisfactory reaction chamber, a cycling chamberinsert, 30, may be employed, as in Fig. 3. This glass or plastic chamber insert 30 acts as linerto prevent reactant from dissolving into the buffer of the gel 32 surrounding the chamber 12.Each insert has walls but no bottom, with the substrate 31 serving as a floor. The outside ofthe chamber insert 30 may be in direct contact with the gel 32, if, for example, the insert 30 isput in place before the polyacrylamide gel is cast.The chamber insert 30 receives the sample and keeps it concentrated. To preventevaporation during the temperature cycling phase, the reactants may be layered with an oil.Alternatively, the chambers may have individual caps which are heated to keep water fromcondensing on the top of the chamber. The chamber insert 30 may be disposable. If it doesl015202530CA 02264961 1999-02-26WO 98/08978 PCT/US97/15056-1]-not have an internal valve or removable wall to release the sample after thermal cycling it cansimply be removed from the gel prior to electrophoresis. A chamber insert material thatdissolves after the reactions are completed could also be used. In the end, the reactionproducts would be conveniently situated in the wells, ready for electrophoretic analysis.An alternative method to release reaction products after the reaction is to use aviscosity trap. This method uses a wax, oil or glycerol which acts as a solid barrier todiffusion at a cool temperature, but which will allow migration of sample when warmed ormelted. By careful positioning of cooling devices, a very small amount of barrier material canbe kept sufficiently cool to prevent the heated reaction products from leaking from thethermocycling region. Upon completion of the reaction, the temperature of the trap is raisedto allow diffusion of the reaction products out of the thermocycling region.Figs. 4 and 5 illustrate alternative designs for the thermocycling region of the sampleprocessing element where the apparatus provides fixed temperature heat zones are used andthe DNA sample migrate from one temperature site to the next, in sequence. The migrationmay be induced by electrophoresis, a thermal capillary pump (see Burns et al. 1991.Microfabricated structures for integrated DNA analysis. Proc. Nat’! Acad. Sci. (USA) 93:5556- 5561), or other methods.In Fig. 4, three separate temperature regions for denaturing, extension and annealingare established by temperature regulating elements 400, 401 and 402. These temperatureregulating elements may be Peltier devices, heat exchangers, or combinations of heat sinks andradiant heaters as disclosed above. A series of reversible electrodes 40, 41, 42 and 43 areemployed to move DNA back and forth between the temperature regions within a bufferreservoir. For example, a sample and accompanying reaction mixture may be initially bedeposited in denaturation region D and treated for an initial denaturation time at denaturationtemperature. Electrodes 40 and 43 (or 15 and 43) are then activated to cause the DNA in thesample to migrate from the denaturation region D to the annealing region A, after which timethe electric field is either turned off or electrodes 42 and 43 are turned on with oscillatingpolarity to maintain it within the region. The DNA is allowed to rest in the annealing region Afor a period of time corresponding to the desired annealing time, after which time electrodes41 and 43 are activated to cause the annealed DNA to migrate to the extension region B andthen turned off. After the desired period of time at the extension temperature, electrodes 401015202530CA 02264961 1999-02-26WO 98/08978 PCT/US97/ 15056-12-and 42 are activated to cause the DNA to migrate back to the denaturation region D. Thiscycle of activating and deactivating electrode pairs is repeated for as many cycles as isnecessary to produce a detectable amount of sequencing fragments. Then electrodes 15 and16 are activated to cause the sequencing fragments to migrate through the separation region102, to the detection region 103.As an alternative to the use of electric fields to move DNA from one temperatureregion to another, DNA can be bound to magnetic beads, such as Dynal beads, and movedfrom one temperature region to the next by an electromagnet. Thus, a denaturedDNA—containing sample is loaded in an annealing region, and allowed to hybridize withprimers. An electromagnet is then turned on to move the DNA to the extension region.Depending on the distances involved, the strength of the magnetic field and the weight of thebeads, the electromagnet may be located under the extension region, or may be a "moving"magnet which starts at the annealing region and moves to the extension region. "Moving" inthis case can refer to physical movement of an electromagnet from one region to the next, orcan be simulated through the use of several magnets which are activated in sequence to createthe same effect.Once the DNA is in the extension region, the electro- magnet may be switched off ormaintained, at a low field strength to limit diffusion of the DNA. At this site the hybridizedprimers and template are exposed to all reagents, including enzymes, that are needed forprimer extension/chain termination. The enzymes may be linked to a solid support and fixed atthe extension site to prevent diffusion in the sample processing element. After sufficient timein the extension region, the DNA is magnetically transported to the denaturing region toseparate the DNA strands. After sufficient time to complete denaturation, the separatedstrands are magnetically transported back to the annealing region. A fresh supply of somereagents may be required to allow for continued reactions. These reagents may be supplied bya continuous drip of fresh reagents.It is noted that only the DNA needs to be treated to the temperature cycling, not theother reaction components. Thus, reaction components, particularly enzymes can beimmobilized within the temperature region where they are needed or added on an as neededbasis to the region where they are consumed.1015202530CA 02264961 1999-02-26WO 98/08978 PCT/U S97/ 15056-13-Fig. 5 shows a variation of the apparatus shown in Fig. 4 in which one circular array ofheat pads are disposed within the thermocycling region 101 for each sample. Each heatingzone D (denaturation), A (annealing) and E (extension) is separately heated and maintainedwithin +/— 0.5 °C. Aqueous buffer covers the entire unit. Again, some reagents maybeimmobilized or added to specific regions during the thermocycling. The reaction mixture isloaded and contained over the heat pads in the thermocycling zone 101, and DNA is drawntowards the denaturation zone D where the temperature is maintained at the denaturationtemperature (i.e., 94°C) by activation of electrodes 501/502 and 505/503. After the desiredamount of time, electrodes 502 and 503, are activated to draw the sample to the annealingregion A. After annealing, electrodes 504 and 505, are activated to draw the DNA to theextension phase temperature region E. Finally, the cycle is completed by activating electrodes506 and 501, to draw the DNA to the denaturing region D of the device. After sufficienttemperature cycles, the sample is denatured one last time, then separated through theseparation matrix upon the activation of electrodes 15 and 16.In the embodiments shown in Figs. 4 and 5, it is only necessary that polymeraseenzyme by present in the extension region B. Thus, a non-thermostable polymerase enzymecan be used if it can be successfully contained within the extension region E. This can beaccomplished by immobilization of the enzyme in the extension region or by placingsemipermeable membranes which restrict the passage of polymerase enzyme but not therelatively smaller DNA molecules around the extension region E.Figs. 6 and 7 shows a further embodiment of the sample processing element of theinvention in which the thermocycling region is a discrete thermocycling and concentrationmanifold 60 which is separable from rather than integral with the gel portion of the sampleprocessing element. The manifold 60 is made up of an array of individual thermocycling andsample concentrating chambers 70, a single one of which is shown in Fig. 7.The manifold 60 has a temperature regulating element 61 disposed in contact therewithfor regulating the temperature of materials within the thermocycling and sample concentratingchambers 70. The manifold 60 fits directly onto the top of an electrophoresis gel, for examplea Visible Genetics Inc. Micr0Ce1TM Cassette, (50 micron thick electrophoresis slab gel) of thetype described in US Patent Application No. 08/332,557 and lntemational Patent PublicationNo. W096/13717, which are incorporated herein by reference. This gel has a top substrate 61l015202530CA 02264961 1999-02-26WO 98/08978 PCT/U S97/ 15056-14-and a bottom substrate 62 surrounding a very thin gel 63. The top substrate 61 has a freebeveled edge 64 which receives the manifold 60.The manifold 60 is suitably made of a thermally conductive material to facilitatetemperature regulation. The manifold may be disposable, or it may include disposable inserts.A sequencing reaction mixture is loaded into each chamber 201 of the manifold 60,and the temperature is then cycled as required to produce sequencing fragments. Theamplified DNA in the sample is then electrophoretically concentrated and loaded onto the gel63.As shown in Fig. 7, each sample concentrating and loading chamber consists of a largerectangular channel 201 which functions as the thermocycling chamber attached at right anglesto a second smaller rectangular channel 202. The upper face of the large rectangular channel201 is open, and receives a volume (for example 100 nL of unconcentrated sample containinga DNA mixture to be separated. The lower face of the smaller rectangular channel 202 is alsoopen and releases the concentrated sample (approx 1 pL) into one of the fiinctional channelsof the DNA sequencing gel 63. There is an unrestricted passageway between the large andsmall channels, to allow sample to flow between them, at a time after a first concentration stepand before a second concentration step.In the first concentration step, sample loaded into the top of the large channel 201 iselectrophoresed using a field generated between electrodes 205a and 205b. The DNA iscollected on a semipermeable membrane 204a which has a molecular weight cutoff lowenough to prevent passage of the DNA but which permits passage of the solvent from thesample, thereby effecting a first concentration of the sample on the semi-permeable membrane204a. Next, a second set of electrodes 206a and 206b are turned on to generate cause theconcentrated sample to migrate in a direction perpendicular to the original migration from thesemipermeable membrane 204a into the small channel 202. A second semipermeablemembrane 204b retains sample within the small channel 202 while permitting passage ofsolvent. Finally, a third electrode set 207a and 207b is used to electrophorese thedoubly-concentrated sample from the small channel 202 into one of the DNA sequencing gel63. In a variation of this device, a valve can be used in lieu of the electric fields to dispensethe fragments onto the gel, but this device does not provide the reduction in sample volume ofthe device shown in Fig. 7.1015202530CA 02264961 1999-02-26WO 98/08978 PCTIUS97/15056-15-Fig. 8 illustrates an alternative embodiment where the manifold 60 is thermal cycledseparately from the gel cassette in a separate heating/cooling block 802, and then clipped ontothe gel cassette 800 immediately prior to loading into the electrophoresis gel, 63. This designprevents the cassette itself from being exposed to thermal cycling conditions, which may provedetrimental to the effectiveness of the sample processing element.Fig. 9 shows an alternative design for a thermocycling chamber which can be used inplaco of or in combination with the loading/concentration manifold of Fig. 7. A capillary tube91 is filled partway with a viscous liquid 92a such as 50% glycerol or 50% sucrose in buffer,then with the PCR solution 93 containing primers, buffer, dNTPs, genomic DNA substrate,ddNTPs and ThermoSequenaseTM enzyme, and then again with further buffered viscous liquid,92b. The diameter of the capillary is small enough that surface tension holds the differentliquid layers without mixing. The two viscous layers, when brought to a low temperature, willact as a viscosity trap 81, to confine the PCR to a small volume. The capillary is placed snuglywithin a channel in a thermal cycling system, 94, such as a block or fluid based heating/cooling system. The ends of the capillary are inserted into buffer chambers 95 to.preventevaporation. The thermocycling system is designed to maintain a temperature ofapproximately 0°C at the two viscosity traps, 92a and 92b, and also to provide temperaturecycling (e.g. 94—55—72°C) at the central PCR solution, 93.After thermal cycling, the capillary is removed from the thermal cycling system and thecontents loaded onto a separation matrix. For example, the capillary can be inserted through ahole or notch in the top substrate of an electrophoresis gel holder, or can be expelled intoconventional sample loading slots of an electrophoresis gel, or can be expelled into a chamberof a loader/concentrator of the type shown in Fig. 7. To expel the sample, the viscosity trapis first opened by warming the capillary. DNA can be moved out of the capillary and onto theseparation matrix using an electric field, or (particularly in the case where the loader is used)can be simply expel led along with the trap components using mild air pressure.In each of the Fig. 6-8 designs and in a gel loaded from the thermocycler chamber ofFig. 9, the reaction mixture may optionally be drawn through a denaturing "loading buffer"just prior to its entrance in the gel, as illustrated in Fig. 10. The entrance to gel 901, betweenthe substrates 902, is layered with a thin wash of denaturing/ stop buffer (50-100 mMformamide, plus dyes, etc), 903. The lower end of the thermocycling and concentrating- 0,20152530CA 02264961 1999-02-26-16-chamber 60 is placed in the buffer 903 leaving a short gap for sample to traverse between thedevice and the gel. 901. Electrodes 904 and 905 which are printed on the inside face of thesubstrate 902 can be switched on to draw the sample through the buffer layer.The detection region of the sample processing element of the invention may simply be acontinuous portion of the separation region, distinguished only by its function. If desired,however, the detection region can have structure specifically adapted to facilitate detection ofthe separated sequencing fragments as they pass through the detection region. Thus, forexample, as disclosed in U.S. Patent No. 5,618,398, which is incorporated herein by reference,one substrate can be made with a thin region localized in the detection region to create awindow for monitoring the detection region with decreased interference from the substrate.Such substrates can be formed by molding a contiguous substrate into the desired shape, or byaffixing blocks of thicker materials onto a continuous thin substrate.: In the latter case, theblocks 71 of thicker material may also be formed from absorbing, non-fluorescing materials tofurther reduce background fluorescence.The apparatus and sample processing element of the invention are utilized in themethod of the invention to sequence DNA, for example the DNA found in natural abundanceDNA samples. In accordance with this method, a therrnostable polymerase enzyme whichincorporates dideoxynucleotides into an extending polynucleotide at a rate which is no lessthan about 0.4 times the rate of incorporation of deoxynucleotides, a natural abundanceDNA-containing sample, two primers flanking the region of DNA to be sequenced, each primerbinding to a different strand of duplex DNA, and other reagents for performing an enzyme-catalyzed primer extension reaction are combined in the therrnocycling region of a sampleprocessing element. The sample processing element is then placed in the apparatus of theinvention and processed through sufficient cycles to produce a detectable amount ofsequencing fragments. An electric field is then generated to cause the sequencing fragments tomigrate through the separation region of the sample processing element, arid thereby beseparate into discrete bands on the basis of the size of the fragments. These bands are thendetected as they pass through the detection region.A key factor in successfully performing the method of the invention is the utilization ofThermosequenasem or a comparable enzyme as the thermostable polymerase in the reactionmixture. Such an enzyme is characterized by a high affinity for incorporating dideoxynucleo-AMENDED SHEET1015202530CA 02264961 1999-02-26WO 98/08978 PCT/U S97/ 15056-17-tides into the extending nucleotide chain. Thus, for example, ThermoSequenaseTM is knownto favor the incorporation of dideoxynucleotides. In general, for purposes of the presentinvention, the polymerase used should be one which incorporates dideoxynucleotides into anextending nucleic acid polymer at a rate which is no less than about 0.4 times the rate ofincorporation of deoxynucleotides.Figs. 1 1A, 11B and 12 illustrate the importance of this characteristic of the polymeraseenzyme employed. Figs. 11A and 12 shows a sequencing data trace for an actual patientsample of genomic DNA which was obtained using ThermoSequenaseTM and primers effectiveto amplify exon 2 of the Von Hippel-Lindau gene in a process according to the invention.Large, well-defined peaks corresponding to the termination fragments were obtained whichmade sequence evaluation of the sample very straight- forward. In addition, the peaks forhomozygous peaks are all approximately the same size, and are readily distinguishable frompeaks for heterozygous locations. This result was obtained performing the test in a singlereaction vessel, with a single unaugmented reaction mixture, in a total of 45 thermal cycles.Comparable results could be obtained using fewer reasctions cycles, for example 35 cycles.In contrast, Fig. llB shows the trace obtained when a combination of Vent andSequithermm was used instead of Thcrmosequenasem for a total of 45 cycles. In this trace,the peaks for the termination fragments are much smaller and less well defined. Furthermore,the peaks are quite variable in height and did not permit identification of heterozygous peaksbased on peak height. Performing the same experiment using Taq polymerase resulted in adata trace that contained no usable peaks.In actual practice, it has been found that useful results are obtained withThermosequenasem when the reaction is run for 35 to 45 cycles, using a dideoxyzdeoxy moleratio of 1:100 to 1:300. Thus, in general it can be expected that mole ratios of 1:50 to 1:500will yield acceptable results. Specific optimum levels for other enzymes found to have theappropriate affinity of incorporating dideoxynucleotides can be identified by routineoptimization using the Thermosequenasem values as a starting point.In the method of the present invention, the two primers used directly produce thesequencing fragments which are analyzed to determine the sequence of the DNA in thesample. Accordingly, at least one of the primers is advantageously labeled with a detectablelabel such as a radiolabel, a fluorophore, a chromophore, a fluorogenic or chromogenic label,101520CA 02264961 1999-02-26WO 98/08978 PCTlUS97l15056-13-or any other label which can facilitate the detection of the sequencing fragments produced inthe reaction. Some full length product (the product spanning from one primer to the other)will also be produced and will be detected during sequencing and may be a substantial bandrelative to any of the individual truncation products. To avoid losing information due to thesize of this band, it may be advantageous to use relatively long primers, for example a 20-25mer such that the difference in length between the full length product and the longest possibletruncation product will be 21 to 26 bases.It may also be advantageous to label both primers used in the method of the invention.For example, the second primer can be labeled with a second detectable label, preferablydifferent in characteristics from the first label. For example, the primers can be labeled withtwo different fluorophores as in the process described by Wiemann et al., "SimultaneousOn-Line DNA Sequencing on Both Stands with Two Fluorescent Dyes," Anal, Biochem 224-1 17-121 (1995). Analysis of the fragments labeled with the two different labels can beaccomplished by loading aliquots of the reaction mixture onto two different electrophoresislanes which are evaluated for different label types or by loading the product mixture onto onelane in a multi-dye sequencer which has the ability to evaluate several labels in a singleinstrument.One of the important characteristics of the present invention is the fact that it permitsconversion of natural abundance DNA to a sequencing product mixture in a single set ofthermocycling reactions without modification of or addition to the reagents present in thereaction mixture. Natural abundance DNA can be prepared from blood or tissue samples byany of a number of techniques, including salt precipitation or standard SDS-proteinaseK-phenol extraction. Natural abundance DNA can also be prepared using kits, for examplethe Gentra Pure Gene DNA Isolation Kit.

Claims

1. An apparatus for processing samples containing DNA to produce a sequencing fragment mixture and for evaluating the fragment mixture comprising (a) a sample processing element comprising:
a thermocycling region have one or more chambers for receiving a DNA
sequencing reaction mixture and forming sequencing fragments therefrom;
a separation region comprising a separation matrix for separating the sequencing fragments formed in the thermocycling regions; and a detection region for detection of the separated sequencing fragments;
(b) means for regulating the temperature in the thermocycling region of the sample processing element to provide a plurality of thermal cycles, each cycle including at least a denaturation phase and an extension phase;
(c) means for applying an electric field to the separation region of a sample processing apparatus placed within a holder to cause polynucleotide sequencing fragments to migrate through the separation region from the thermocycling region to the detection region;
and (d) means for detecting polynucleotide fragments within the detection region of the sample processing apparatus placed within the holder, characterized in that the means for regulating the temperature comprises at least three temperature regulating elements, a first temperature regulating element for maintaining a first portion of the thermocycling region at a denaturation temperature; a second temperature regulating element for maintaining a second portion of the thermocycling region at an annealing temperature, and a third temperature regulating element for maintaining a third portion of the thermocycling region at an extension temperature.

2. The apparatus according to claim 1, wherein the means for regulating the temperature is a Peltier device disposed in thermal contact with the thermocycling region of the sample processing element.

3. The apparatus according to claim 1, wherein the means for regulating the temperature comprises a heat sink maintained at a temperature below the desired temperature of the thermocycling region and a source of radiant heat.

6. The apparatus according to any of claims 1 to 3, wherein the means for detecting polynucleotide fragments within the detection region comprises a source of radiation for exciting a fluorescent label on the sequencing fragments and a detector for detecting fluorescent emission from the sequencing fragments.

7. The apparatus according to claim 6, wherein the means for detecting polynucleotide sequencing fragments within the detection region comprises a plurality of detectors.

8. The apparatus according to claim 6, wherein the means for detecting polynucleotide sequencing fragments comprises means for splitting radiation from the excitation source into a plurality of excitation beamlets.

9. The apparatus according to claim 8, wherein the means for splitting radiation from the excitation source into a plurality of excitation beamlets is a spot array generation grating.

10. The apparatus according to any of claims 1-3 or 6-9, wherein the means for applying an electric field is a pail of solution electrodes.

11. The apparatus according to any of claims 1-3 or 6-10, wherein the separation region comprises a polyacrylamide electrophoresis gel, and wherein the chamber is a well formed in the gel near a first end thereof.

12. The apparatus according to claim 11, further comprising a removable liner disposed within the well for separating the DNA sequencing reaction mixture from the gel during the formation of the sequencing fragments.

13. The apparatus according to any of claims 1-3 or 6-10, wherein the thermocycling region comprises a manifold formed from a plurality of chambers arranged in a line, each of said chambers in the manifold having a top opening for receiving the sequencing reaction mixture and a bottom opening for transferring sequencing reaction fragments formed in the separation region.

14. The apparatus according to claim 13, wherein each chamber of the manifold has connected thereto a concentrating device for concentrating the sequencing fragments formed therein prior to transferring them to the separation region.

15. The apparatus according to claim 13 or 14, wherein the manifold is separable from the separation region.

16. A method for sequencing a selected region within DNA molecules in a natural abundance sample comprising the steps of (a) combining the natural abundance sample with a thermostable polymerase enzyme which incorporates dideoxynucleotides into an extending polynucleotide at a rate which is no less than about 0.4 times the rate of incorporation of deoxynucleotides, two primers flanking the selected region of DNA to be sequences, each primer binding to a different strand of duplex DNA, nucleotide triphosphate feedstocks and a type of chain terminating nucleotide triphosphate to form a sequencing reaction mixture;
(b) placing the sequencing reaction mixture in an apparatus according to any of claims 1-3 or 6-15;
(c) processing the sequencing reaction mixture through a plurality of thermal cycles, each cycle including at least a denaturation phase and an extension phase to form sequencing fragments;

(d) separating the sequencing fragments; and (e) detecting the separated sequencing fragments.

20. The method according to claim 16, wherein the thermocycling region comprises a manifold formed from a plurality of separate chambers arranged in line, and wherein each chamber of the manifold has connected thereto a concentrating device for concentrating the sequencing fragments formed therein prior to transferring them to the separation region.